Protein A chromatography is widely employed for the preparative purification of proteins possessing a heavy chain Fc region comprising the CH2 and CH3 domains of an immunoglobulin molecule. (Vola et al. Cell Biophys. 24-25: 27-36, 1994; Gagnon, Protein A affinity chromatography, In: Purification tools for monoclonal antibodies, 1996, Validated Biosystems, Tucson, Ariz., 1996; Aybay and Imir, J. Immunol. Methods 233(1-2): 77-81, 2000; Ford et al., J. Chromatogr. B 754: 427-435, 2001; Fahrner et al, Biotechnology and Genetic Engineering News, 18: 301-327, 2001). Such Fc proteins include antibodies, particularly monoclonal antibodies, as well as Fc fusion proteins. Such proteins are typically produced by mammalian or bacterial cells engineered to express the desired recombinant protein, intracellularly or directly, into the culture medium or intracellularly. Purification of the expressed proteins typically begins with either collecting the cell culture medium to harvest extracellularly expressed protein or harvesting and lysing the host cells to release intracellularly expressed protein. This host cell “harvest medium” contains not only the protein of interest but also DNA, RNA, and protein contaminants from the host cell that must be separated from the desired protein. These host cell contaminants may be separated by various chromatographic methods based on their charge, size or hydrophobicity. The affinity of immunoglobulin heavy chain Fc regions for the IgG binding domains of Protein A allows for the direct capture and purification of such Fc proteins from complex host cell harvest medium that may contain many different host cell contaminants. Despite the high specificity of the Fc region for Protein A, host cell protein contaminants may still be present at varying levels in the final column eluate, thereby reducing the purity of the of the final protein product. To be useful for human therapeutic purposes, protein products must be separated from all of the extraneous media components and cell by-products, creating a need for purification methods that can maintain product yield while reducing host cell contaminant levels.
Typically Protein A affinity chromatography consists of a column comprising Protein A immobilized on to a solid support and equilibrated to a neutral pH. Cell culture harvest medium containing the desired protein product in addition to host cell contaminants is loaded directly onto the Protein A column followed by a preliminary wash with an equilibration buffer at an intermediate pH to remove any host cell contaminants that were not bound to the protein product of interest or the Protein A matrix. This is followed by an intermediate wash step to remove any bound host cell protein contaminants. As described below, such contaminants may bind to the Protein A matrix and/or to the protein of interest. The formulation of the intermediate wash buffer is typically similar to the elution buffer, except for having a more intermediate pH in place of the lower pH of the elution buffer. Following the intermediate wash step the protein product is then eluted from the Protein A column using an elution buffer.
For large-scale purification much effort is placed on optimizing the formulations of wash and elution buffers to maximize product yield. However, in a production situation where many different protein products are being purified at the same time, developing a unique wash buffer for each individual protein product requires significant time and resources to screen various buffer formulations to determine an appropriate wash buffer for each particular protein product. A “generic” intermediate wash buffer that could be used effectively with different types of proteins would be useful and desirable. A common path taken when designing wash buffers is to mimic elution buffer formulations but within an intermediate pH range. However, since elution buffer formulations are designed to maximize the recovery of a particular protein product these formulations are typically protein-specific and not easily transferred from one protein to another and typically are at lower pH, increasing the possibility of loss of product during the wash step due to weakening of the interaction between the protein product and the Protein A. Therefore, the formulation of the intermediate wash buffer should maintain a balance between contaminant removal and loss of protein product. Thus, it would be desirable to develop a “generic” wash buffer that could be used over a broad range of protein products, such as monoclonal antibodies and Fc-fusion proteins, that would maximize removal of host cell protein contaminants while minimizing loss of protein product during Protein A affinity chromatography.
The present invention provides a method of protein purification using such wash buffer formulations.